THE EFFECTS OF REPORTED CA-2+ SENSITIZERS ON THE RATES OF CA-2+ RELEASE FROM CARDIAC TROPONIN-C AND THE TROPONIN-TROPOMYOSIN COMPLEX

The calcium sensitivity of force production of cardiac muscle fibres is altered by certain drugs. The sites of action of three such compounds (pimobendan, sulmazole, isomazole) within the myofibril have been investigated. Calmodulin antagonists perhexilene and bepridil, which have been shown to alter the calcium dependence of myofibrillar ATPase activity and oxmetidine, and H2-receptor antagonist which binds to calmodulin, were also studied. The rates of dissociation of calcium from both the regulatory and high affinity sites on bovine isolated cardiac troponin C (cTnC) were measured in a stopped-flow fluorimeter. The rates of dissociation were found to be 136.5 ± 16 s-1 and 1.3 ± 0.20 s-1 (mean ± s.e. mean, n = 11 determinations; conditions: 100 mM KCl 10 mM MOPS, 3 mM MgCl2, 0.1 mM dithriothreitol, pH 7.0, 15°C). Sulmazole, isomazole and perhexiline (final concentration of 50 μM) had no effect on the rate of Ca2+ dissociation from the regulatory Ca2+ site, indicating that these compounds do not act on cTnC directly. The rate of dissociation of Ca2+ from the regulatory site was slightly reduced (~20%) by pimobendan (50 and 100 μM) and was somewhat increased by oxmetidine (28% at 100 μM). Bepridil (25 μM) reduced the rate of dissociation by 50%, indicating a direct effect of bepridil on TnC. Sulmazole, isomazole, perhexiline, pimobendan (50 μM) and bepridil (25 μM) were without effect on the rate of dissociation of Ca2+ from the high affinity Ca2+/Mg2+ sites. Oxmetidine caused 24% decrease in the rate of Ca2+ dissociation from these sites. The rate of dissociation of Ca2+ from the regulatory site on the complex of troponin-tropomyosin (TnTm) was measured. Sulmazole and pimobendan (50 μM) were without effect on the rate of dissociation of Ca2+ from the regulatory site in the protein complex, and isomazole (50 μM) caused only a slight reduction (23%). Perhexiline (50 μM) or bepridil (10 μM) reduced the rate of Ca2+ dissociation by about 50%. The rate of dissociation of Ca2+ from the high affinity Ca2+/Mg2+ sites was not altered by sulmazole, isomazole, or pimobendan (50 μM), but was decreased ~35% by perhexiline (50 μM) or bepridil (10 μM).